In the specification, footnotes used are to the following references, which are hereby incorporated by reference into the specification.
1. Edwards, W. H. and Mulherin, J. L. The role of graft material in femorotibial bypass grafts Ann. Surg. 191:721, 1980.
2. Herring, M., Gardner, A., Glover, J., Seeding human arterial prosthesis with mechanically derived endothelium. The detrimental effect of smoking, J. Vasc. Surg., 1984, 1 (2), 279-289.
3. Williams, S., Jarrell, B., Friend, L., Radomski, J., Carabasi, R., Koolpe, E., Mueller, S., Thornton, S., Marinucci, T., and Levine, E. Adult human endothelial cell compatibility with prosthetic graft material. J. Surg. Res. 38:618, 1985.
4. Jarrell, B., Williams, S., Solomon, L., Speicher, L., Koolpe, E., Radomski, J., Carabasi, R., Greener, D., and Rosato, F. Use of an endothelial monolayer upon a vascular graft prior to implantation: temporal dynamics and compatibility with the operating room. Ann. Surg, Vol. 203 No. 6, 671-678, 1986.
5. Jarrell, B., Williams, S., Stokes, G., Hubbard, F., Carabasi, R., Koolpe, E., Greener, D., Pratt, K., Radomski, J., Speicher, L., and Moritz, M. Use of freshly isolated capillary endothelial cells for the immediate establishment of a monolayer on a vascular graft at surgery. Surgery Vol. 100, No. 2, 392-399, 1986.
6. Radomski, J., Jarrell, B., Williams, S., Koolpe, E., Greener, D., and Carabasi, R. Initial adherence of human capillary endothelial cells to Dacron, J. Surg. Res. Vol. 42, 133-140, 1987.
7. Baker, K.S., Williams, S., Jarrell, B., Koolpe, E., and Levine, E. Endothelialization of human collagen surfaces with human adult endothelial cells. Amer. J. Surg. 150:197, 1985.
8. Durand, R., and Olive, P. Cytotoxicity, mutagenicity and DNA damage by Hoechst 33342 J. Histocgem Cytochem. 30:111, 1982.
9. Williams, S., Sasaki, A., Matthews, M., and Wagner, R. Quantitative determination of deoxyribonucleic acid from cells collected on filters. Anal Biochem. 107:17, 1980.
10. Greenspan, P., Mayer, E., and Fowler, S., Nile Red: a selective fluorescent stain for intracellular lipid deposits. J. Cell. Biol. 100:965, 1985.
11. Johnson, L., Walsh. M., and Chen, L. Localization of mitochondria in living cells with rhodamine 123. P.N.A.S.. U.S.A. 77:990, 1980.
12. Jarrell, B., Shapiro, S., Williams, S., Carabasi, R., Levine, E., Mueller, S., and Thornton, S. Human adult endothelial cell growth in culture. J. Vasc. Surg. 6:757, 1984.
13. Thornton, S., Mueller, S., and Levine, E. Human endothelial cells: cloning and long-term serial cultivation employing heparin. Science 222:623, 1983.
14. Jaffe, E. A., Nachman, R., Becker, C., and Minick, C. Culture of human endothelial cells derived from umbilical veins. J. Clin. Invest 52:2745, 1973.
15. Arndt-Jovin, D., and Jovin, T. Analysis and sorting of living cells according to deoxyribonucleic acid content. J. Histochem. Cytochem 25:585, 1977.
16. Johnson, L., Summerhayes, I., and Chen, L. Decreased uptake and retention of rhodamine 123 by mitochondria in a feline sarcoma virus-transformed Mink cells. Cell, 28:7, 1982.
17. Chen, L., Summerhayes, I., Johnson, L., Walsh, M., Bernal, S., Lampidis, T, Probing metochondria in living cells with rhodamine 123. Cold Spring Harbor Symposium Quant. Biol. 46:141, 1982.
18. Ziegler, M. and Davidson, R. Elimination of mitochondrial elements and improved viability in hybrid cells. Somatic Cell Genetics, 7:73, 1981.
Graham et al. Surgery 91(5):550-559 (1982) teach harvesting large vessel endothelial cells from dog external jugular veins, culturing the harvested cells, and implanting the seeded grafts in dogs for several weeks. The grafts are then removed and graft segments prepared for study by SEM, TEM and light microscopy. Representative 1.0 sq. cm specimens of the grafts are prepared for AgNO.sub.3 staining by rinsing in a 5% glucose solution for 5 minutes,
then placing them in a 0.5% AgNO.sub.3 solution in direct sunlight until discoloration becomes apparent, rinsing and then fixing in formalin for microscopic examination. Graham et al. also teach fixing graft segments in glutaraldehyde, dehydrating in ethanol, and embedding or critical-point drying for scanning electron microscope (SEM) observation. For light microscopy Graham et al. teach staining with methylene blue. For transmission electron microscopy (TEM), Graham et al. teach fixing in glutaraldehyde and embedding in Epon. From these animal studies, Graham et al. seek to test the efficacy of endothelial cell seeding on excised ePTFE grafts.
Herring et al. Biol. Abstr. 69(3):1559 (1979) teaches seeding a graft with endothelial cells, implanting the grafts in dogs and then removing the grafts 6 weeks later for examination. F VIII-RA is used to stain the cells lining the excised graft. Silver nitrate Hautchen and EM preparations show a lining pattern characteristic of vascular endothelium.
Summerhayes et al. Proc. Natl. Acad. Sci. USA 79:5292-5296 (1982) teach Rhodamine 123 as a mitochondria-specific fluorescent probe in living cells, particularly useful for studying cancer cells for either diagnostic or chemotherapeutic monitoring purposes. In Table 1, on page 5294, Summerhayes et al. show Rhodamine retention of various normal cell types, including human aorta and bovine aorta endothelial cells. This data shows the unusual retention of Rhodomine 123 by mitochondria in muscle or carcinoma cells.
Williams et al. Anal. Biochem. 107:17-20 (1980) teach a modified fluorescence assay for the quantitation of cellular DNA extracted from mammalian cells collected on cellulose triacetate membrane filters using mithramycin.
Chen et al. Cold Spring Harbor Symposia Quant. Biol. 46:141-155 (1985) teach using Rhodamine 123 for visualizing mitochondria in live cells and as a measure of cell viability after cytotoxic drug exposure. According to Chen et al.'s methods cell aliquots are stained and then analyzed by flow cytometry. Some mitochondria prepared according to Chen et al.'s methods can also be observed with phase-contrast optics.
Greenspan et al. J. Cell Biol. Abstract No. 9629, 100(3) 965-974 (1985) teach using Nile red as a fluorescent lipid stain for fluorescence microscopy and flow cytometry.
None of these references teach or suggest using a non-toxic fluorescent dye to evaluate the extent of microvascular endothelial cell coverage or the extent of cell-to-cell interactions on a prosthetic surface prior to implantation.
The replacement of damaged blood vessels with prosthetic vascular grafts has become a feasible surgical option in recent years. While the use of large lumen grafts has met with considerable success, small lumen grafts often become occluded due to the thrombogenic nature of the materials. Attempts have been made to reduce the thrombogenicity of the grafts by seeding with endothelial cells prior to implantation. While this method has proved feasible in animal models, similar attempts using human endothelial cells have not demonstrated improved patency (2).
A procedure for attaching human endothelial cells to vascular graft material in vitro has been recently developed (3). It is now possible to establish an endothelial cell monolayer on a graft after a 1 to 2 hour incubation period (4-7). Before this technique can be performed in the operating room, however, a simple method to determine the completeness of the seeding process is necessary. Present methods to visualize endothelial cells on graft material involve fixation procedures which irreversibly damage the cells. Accordingly, there remains a need for a relatively rapid, non-destructive, method of determining the degree of confluence of seeded endothelial cells on a prosthetic surface.